The present invention relates generally to the treatment of one or more fungal diseases and more particularly, to the discovery that selected structural modifications of dolastatin 10 are remarkably useful as fungicides.
The elucidation and isolation of dolastatin 10 is described in U.S. Pat. No. 4,816,444 which issued to G. R. Pettit et al., on Mar. 28, 1989 while early efforts to synthesize dolastatin 10 and develop certain modifications thereof are described in U.S. Pat. Nos. 4,978,744; 5,410,024; 5,504,191; 5,521,284; 5,530,097; 5,599,902; 5,635,483; 5,663,149; and 5,665,860, all of which issued to G. R. Pettit et al. between 1990 and 1997. The general background information from each of the above cited U.S. patents is incorporated herein by this reference thereto.
Dolastatin 10 is a linear peptide containing four unusual amino acids (compound 1a, FIG. 1) and was originally isolated from the Indian Ocean sea hare Dolabella auricularia (See: Pettit et al., The isolation and structure of a remarkable marine animal antineoplastic constituent: Dolastatin 10, J Am Chem Soc 1987, 109: 6883-85). The synthesis of dolastatin 10 (Pettit et al., The absolute configuration and synthesis of natural (xe2x88x92)-dolastatin 10, J Am Chem 1 Soc 1989, 111: 5463-65; Pettit et al., Dolastatins 24. Synthesis of (xe2x88x92)-dolastatin 10. X-ray molecular structure of N,N-dimethylvalyl-valyl-dolaisoleuinetert-butyl ester, J Chem Soc, Perkin Trans 1 1996, 859-63) facilitated detailed investigation of its powerful antineoplastic activities (see, Pettit, The dolastatins., In: Herz et al., (eds.) Progress in the Chemistry of Organic Natural Products, 70th edn. New York: Springer-Verlag, 1997: 1-79), culminating in the initiation of Phase I cancer clinical trials in 1995. The sea hare-derived peptide dolastatin 10 is currently in phase I and phase II cancer clinical trials. In mammalian cells, the intracellular target of dolastatin 10 is tubulin. The peptide inhibits microtubule assembly and tubulin-dependent GTP binding (Bai et al., Dolastatin 10, a powerful cytostatic peptide derived from a marine animal: Inhibition of tubulin polymerization mediated through the vinca alkaloid binding domain, Biochem Pharmacol 1990, 39 (12): 1941-49), and is a noncompetitive inhibitor of vincristine binding to tubulin (Bai et al., Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites, J Biol Chem 1990, 265 (28): 17141-49). Dolastatin 10 causes metaphase arrest in a wide variety of animal and human cancer cell lines, and exhibits impressive activity in murine tumor models (see Pettit, 1997, supra). In addition, dolastatin 10 induces apoptosis in certain human lymphoma cells lines (Beckwith et al., Growth inhibition of human lymphoma cell lines by the marine products, dolastatins 10 and 15, J Nat Cancer Inst 1993, 85 (6): 483-88; Maki et al., The bcl-2 and p53 oncoproteins can be modulated by bryostatin 1 and dolastatins in human diffuse large cell lymphoma, Anti-Cancer Drugs 1995, 6: 392-97). The apoptotic mechanism is apparently unrelated to its antimitotic effects (Beckwith et al., 1993, supra; Maki et al., 1995, supra). In ongoing clinical trials in patients with advanced solid tumors, there is minimal toxicity at doses up to 200 xcexcg/m2 (Tran et al., A Phase I, pharmacokinetic/pharmacodynamic study of dolastatin 10 in adult patients with advanced solid tumors, Proceedings of the American Association for Cancer Research, San Diego, Calif. 1997, #2056; Bagniewski et al., Pharmacokinetics of dolastatin 10 in adult patients with solid tumors, Proceedings of the American Association for Cancer Research, San Diego, Calif. 1997, #1492; McElroy et al., Phase I trial of dolastatin 10 in patients with advanced solid tumors, Proceedings of the American Society of Clinical Oncology, Denver, Colo. 1997, #782).
The tubulin-binding properties discovered for dolastatin 10 prompted a further look to consider whether dolastatin 10 might also be useful as an antifungal agent. The effort resulted in dolastatin 10 and four specific structural modifications thereof (FIG. 1) being identified as potent and specific fungicidals which are specifically active against Cryptococcus neoformans. 
The antifungal spectrum of dolastatin 10 and four structural modifications thereof has been evaluated and is disclosed herein. In broth macrodilution assays, the peptides were fungicidal for ATCC strains and clinical isolates (including fluconazole-resistant strains) of Cryptococcus neoformans, but not for any of the other yeasts or filamentous fungi examined. Specificity for C. neoformans was also demonstrated in the solid-phase disk diffusion assay, and fungicidal activity was confirmed in killing kinetics experiments. The MICs at which 50% and 90% of 19 clinical isolates were inhibited (MIC50, MIC90) by a methyl ester modification were 0.195 xcexcg/ml and 0.39 xcexcg/ml, respectively. The MFC50, (minimum fungicidal concentration) for this peptide was 0.39 xcexcg/ml, and the MFC90 was 0.78 xcexcg/ml. Broth macrodilution minimum inhibitory and minimum fungicidal concentrations for the most potent modification ranged from 0.0975-1.56 xcexcg/ml and 0.0975-6.24 xcexcg/ml, respectively. The minimum inhibitory concentrations (MICs) were nearly identical in the presence of human serum, but increased with lowered pH. These peptides offer untoward promise as potential chemotherapeutics for C. neoformans, a leading cause of infection and mortality in immunocompromised patients.
Accordingly, the prime object of the present invention is to provide new anti-cryptococcal peptides and methods of using same.
Another object of the present invention is to provide the elucidation and identification of dolastatin 10 and structural modifications thereof for use as fungicides.
These and still further objects as shall hereinafter appear are readily fulfilled by the present invention in a remarkably unexpected manner as will be readily discerned fiom the following detailed description of exemplary embodiments thereof especially when read in conjunction with the drawings attached hereto.